PROJECT SUMMARY PROJECT 2 There are currently 120,000 patients in the United States living with multiple myeloma (MM) and a vast majority will die of their disease despite high initial rates of response to recently introduced anti-MM agents. Intensive chemotherapy followed by autologous stem cell transplant (ASCT) can increase complete remission (CR) rates and prolong survival. Nonetheless, MM remains incurable and sequential lines of treatment for each inevitable relapse results in progressively shorter durations of response due to acquired resistance by MM cells to available therapy. Ultimately, almost all patients develop treatment unresponsive MM, which leads to the death of over 11,000 Americans annually. Immunotherapy, using antibodies specific for tumor associated molecules, immune checkpoints, or the adoptive transfer of tumor reactive T cells, has emerged as an effective modality for many cancers, including MM. Adoptive cell therapy with T cells engineered by gene transfer to express synthetic chimeric antigen receptors (CARs) represents a potentially transformative approach for MM. CAR-T cells are not HLA-restricted and unlike T cell receptor directed therapy, can treat all patients with tumors that express the surface molecule recognized by the CAR. This project will explore CARs designed and optimized with specificity for B cell maturation antigen (BCMA) a transmembrane receptor that is expressed on MM. These CARs contain novel features that improve function, enable in vivo tracking, and facilitate CAR-T cell manufacturing. T cells expressing our optimized BCMA CARs are effective against MM in vitro and in preclinical models. This Project (Project 2), a new direction on this grant, proposes a clinical trial of autologous T cells transduced with BCMA CARs containing different costimulatory domains and formulated in a uniform T cell subset composition for patients with MM. The studies will determine whether there are differences in toxicity and function of CAR-T cells expressing CD28/CD3? or 4-1BB/CD3? signaling domains, and identify tumor cell intrinsic and extrinsic mechanisms of MM resistance to ACT. A focus of the proposal is to develop strategies to overcome resistance to ACT and obtain a higher rate of complete remission. We have identified low BCMA expression as a potential barrier to T cell recognition of MM, and preclinical data shows that measures can be taken to increase surface BCMA on MM, while decreasing soluble BCMA levels. Lenalidomide (Len) is a critical component of current MM therapy and preclinical studies are proposed to examine the effects of Len on CAR--T cells to support future clinical trials of CAR--T cells in combination. Overall, Project 2 will determine the safety and efficacy of each of the CAR constructs, and enhance the understanding of how costimulatory signaling affects the fate and function of CAR-T cells while employing preclinical models to prospectively address potential barriers to CAR-T cell efficacy. Findings will inform future clinical trials and advance the field of adoptive cell therapy.